Infection of the respiratory tract by M. pneumoniae is initiated byattachment of the organism to ciliated cells. Attachment of the organism to the respiratory epithelium is a prerequisite to colonization. Attachment is followed by ciliostasis, alteration and loss of cilia, and destruction of the mucosal epithelium. The nature of events leading to the destruction of the respiratory epithelium is not clear. Chandler et al (1980. I & I. 29:1111-1116) demonstrated that a cell free extract of a virulent M. pneumoniae caused ciliostatis of hamsters ciliated epithelium, hemagglutination of red blood cells, and proteolysis of a synthetic tetrapeptide substrate S-2222. An open reading frame 6 (ORF6) coding for a 130 kDa protein, is part of the "P1 adherence operon" of M. pneumoniae (Colman et al. 1990. Gene. 87:91-96). The 40 kDa and 90 kDa protein coded for by the ORF are important in positioning P1 adhesin at the tip structure of the organism. Processing signals for production of the two proteins can not be identified on a nucleic acid level. Our current working hypothesis is that the protease is required to cleave the 130 kDa scaffolding protein. OFR6 will be recloned into phagemid Bluescript and the 130 kDa protein will be labeled radioactively under the T7 polymerase system. The labeled protein will serve as a substrate for digestion by the protease. Two approaches have been taken to study the protease. First, the protease will be isolated from a cell free extract, and its specificity will be studied using protease inhibitors and substrates. Second, a gene coding for a UGA suppressor tRNAtrp was obtained from Michael Yarus (Univ. of Colorado). The gene was recloned into plasmid pRK415 that is compatible with the ColEI replicon plasmids used to construct a chromosomal DNA library from M. pneumoniae strain M129. The library, constructed in a IacIq strain under the Iac promoter, will be induced and screened for production of the protease.